1. Field of the Invention
This invention relates to a method and apparatus for measuring mold growth.
2. Description of Related Art
Mycotoxin contamination of foodstuffs is a common problem impacting the grain, feed, and animal industries. It is known that at least 300 different mycotoxins can contaminate cereal grains and oil seeds. Contamination of these foodstuffs can result in destruction of large quantities of grain. Additionally, since these commodities typically represent a major component of animal feeds, the threat to animal health from mycotoxin contamination is significant. Due to an increased awareness of the potential health hazards associated with mycotoxins, and recent advances in the testing of feedstuffs for the presence of mycotoxins, contamination of grains and feed by these compounds is considered one of the major problems facing the grain and animal industries. Mycotoxin contamination of foodstuffs is the result of uncontrolled growth of certain toxigenic molds. Mycotoxins are highly toxic metabolic by-products, released into the immediate environment as these molds grow. As time proceeds, the molds responsible for the production of the mycotoxins may become non-viable. However, in most cases the mycotoxins remain due to their high chemical stability.
A logical approach to minimizing mycotoxin contamination in foodstuffs is to minimize mold growth. Numerous approaches have been employed by the grain, feed, and animal industries to minimize mold growth in foodstuffs: (1) Proper use of insecticides, fertilizers, and irrigation techniques reduces greatly the probability of mold growth and mycotoxin formation in pre-harvest grain. (2) Early harvest of grain usually leads to minimal mold growth and minimal mycotoxin contamination because mold growth occurs in pre-harvest grains near the end of the growing cycle of the grain. (3) Immediate, rapid and complete drying of harvested grains retards the growth of molds in post-harvest grain. However, the drying procedures must be initiated as soon after harvest as possible, and the final moisture of the grain must be low enough to prevent mold growth. (4) Storage of grain and manufactured feeds in storage facilities that are dry, water-tight, and free of moldy and caked material assists in the prevention of mold growth and mycotoxin contamination in grain or feed stored in these storage containers. (5) Rapid use of manufactured feeds decreases the chance of mold growth during the period between feed manufacture and feed consumption. (6) The use of chemical preservatives minimizes mold growth in grain and feed, thereby minimizing the change of mycotoxin contamination in these commodities. Given the relevance and importance of mold growth to mycotoxin contamination of feedstuffs, it is obvious that an accurate method for measuring mold growth in commodities where the cultural conditions closely mimic practical or "field" conditions is imperative. This is particularly important when the efficacy of chemical preservatives is being investigated. A chemical believed to retard the growth of molds in feed must be supported by evidence of less mold growth in feed when the chemical is used than when it is not.
Unfortunately, accurate measurement of mold growth in an amorphous substrate, such as poultry feed, is much more difficult than the measurement of other (such as bacteria) in feed. Most bacteria and yeasts reproduce as single cells or conglomerates comprised of single cells. Therefore, mixing an appropriate diluent with a sample to be analyzed will result in suspension of the cells in the diluent. The diluent can then be diluted further, and the number of viable bacteria or yeasts (indicative of the degree of microbial growth) can be determined by plating the dilutions on an appropriate medium and counting the resulting bacterial colonies. Molds do not reproduce or grow in this fashion in most agricultural commodities. The growth of molds is characterized initially by the development of mycelium. This early stage of mold growth is not visible to the unaided eye. As the mold continues to grow, this mycelium proliferates and forms a continuous and filamentous network throughout the feed. Associated with this mass is also the development of aerial mycelium which serve to project the reproductive spores above the surface of the feed particle. This mycelial mass often becomes an integral part of the individual particles of the commodity being analyzed. Techniques used for the assessment of bacterial or yeast growth are therefore not suitable for assessment of mold growth.
Perhaps the most traditional method for the estimation of mold growth has been the "mold spore count". The premise of this technique is that "the more mold spores in a commodity, the greater the expected mold growth." Mold spores occur singly or as conglomerates, and therefore can be enumerated in a manner similar to that used for the enumeration of bacteria and yeasts. The technical simplicity and the "assumption" that mold spore concentration is indicative of mold growth are the main reasons for the use of this technique to estimate mold growth in feedstuffs. The fallacy in the use of the mold spore count for estimation of mold growth lies in the fact that "sporulation" by molds and "growth" of molds can be independent biological events: a given mold may grow abundantly in feed, but sporulate sparsely. Enumeration of mold spores in this case would lead to the conclusion that only sparse mold growth has occurred in the substrate, when the opposite would be true. Other molds are known to grow sparsely, but produce abundant spores. In this situation also, enumeration of mold spores would lead to erroneous conclusions.
A fundamental concept in microbiology is that microbial growth (including molds) can be measured indirectly by the disappearance of a substrate or the generation of a by-product as a result of growth of the organism. Respirometry has long been used to measure microbial growth in a closed system, usually by measuring oxygen consumption. The Warburg respirometer has been used previously for this purpose, but is not always sufficient because of several limitations. (1) Oxygen consumption is measured by the detection of small changes in pressure within the system, while carbon dioxide is absorbed by the presence of potassium hydroxide in the growth chamber. (2) Only a measurement of oxygen consumption (disappearance of a substrate associated with microbial growth) is capable with the Warburg respirometer. (3) Additionally, since oxygen consumption is determined by slight changes in pressure within the system, extremely stable temperatures are required. (4) In many cases where the actively growing organisms generate heat, the use of the Warburg respirometer is not suitable. (5) Since the Warburg respirometer is a true "closed system", no provisions can be made for the replenishment of oxygen consumed by the microorganisms. Therefore, as oxygen is depleted from the atmosphere within the respirometer, aerobic organisms (i.e. molds) may be unable to maintain optimum growth in an unrestricted state due to the increasing concentration of carbon dioxide and the decreasing concentration of oxygen. (6) Furthermore, the Warburg respirometer requires manual reading of pressure changes within the "closed system"; frequent and periodic measurements are often not practical.
Recently, a unique respirometer ("MICRO-OXYMAX" 20), Columbus Instruments, Columbus, Ohio) has been developed that permits the simultaneous measurement of oxygen consumption and carbon dioxide generation in a "closed system" (See U.S. Pat. No. 4,947,339 and FIG. 1). The air, in up to 20 chambers, is periodically circulated through highly sensitive oxygen and carbon dioxide sensors and then returned to the chambers. The respirometer measures changes in gas concentrations in the chambers with respect to time. Changes in oxygen and carbon dioxide concentrations, coupled with the volume of the chamber and the time elapsed between measurements, permit the calculation of the rate at which oxygen is consumed and the rate at which carbon dioxide is produced. Additionally, the cumulative consumption of oxygen and cumulative production of carbon dioxide can also be determined. The cumulative measurements are indicative of the growth of the mold on the substrate. The rate measurements can be used to determine the rate of mold growth. One useful feature of this particular respirometer is its capability to be programmed to replace or "refresh" the air in each chamber with room air. During long experiments, the concentrations of oxygen and/or carbon dioxide may change significantly from starting concentrations to the extent that the growth rate of the organisms in the chamber may be affected adversely. The user may, in such cases, choose to configure the respirometer to "refresh" each chamber periodically. This maintains optimum levels of oxygen and carbon dioxide equal to those at the onset of the experiment.
Using a combination of valves and switches, the sensors are repetitively and sequentially connected to each of the 20 incubation chambers at user-determined intervals. A microcomputer with specially designed software is used to control the entire system, including the control of measurement of oxygen and carbon dioxide concentrations, calculation of the results, and printing or saving the results to floppy or hard disk. The system also incorporates facilities to assist in sensor calibration and automatic measurement of incubation chamber volumes and barometric pressure.
The "MICRO-OXYMAX" 20 respirometer employs a very stable, single beam, non-dispersive, infrared carbon dioxide sensor that operates over the range of 0-1% carbon dioxide. The oxygen sensor is electrochemical (fuel cell) and has the capability of measuring directly the percentage of oxygen in the chamber atmosphere.
This apparatus is not completely adequate for situations where moisture content of samples is important. No provision is made in this apparatus to control the temperature of the environment surrounding the chambers. Additionally, no provision is made to control the moisture level of the substrate used within each chamber. In the case of moist poultry feed, as the experiment progresses and the atmosphere within each chamber is sampled, water is removed from the atmosphere by the drying column (shown on the left of the system in FIG. 1 is a pump) prior to analysis for specific gas concentrations. During repetitive sampling of the atmosphere within each chamber over time, the samples tend to dehydrate. This dehydration interferes with the normal growth of the mold and often leads to incorrect conclusions.